Influence of Sporulation Medium Composition on Transcription of ger Operons and the Germination Response of Spores of Bacillus cereus ATCC 14579

Bacillus cereus ATCC 14579 endospores were produced in Y1 medium,a nutrient-rich, chemically defined sporulation medium, andin modified G medium, containing low amounts of nutrients. Theaverage transcription level of the seven ger operons per cellwas 3.5 times higher in Y1 medium, and the spores grown in thismedium showed an enhanced germination response.

INTRODUCTION

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Introduction

Bacillus cereus is a gram-positive, rod-shaped food pathogenthat can sporulate under nutrient-limiting conditions. The conditionsunder which sporulation takes place determine spore characteristicssuch as wet-heat resistance and germination properties (7, 18,22, 26). Once formed, spores can return to vegetative growthby an irreversible process called germination (19, 21, 24).Germination receptors, which are located in the inner membraneof the spore, play a crucial role in the initiation of germination,as spores lacking these receptors are strongly impaired in theirresponses to germinant molecules (20). The signaling and activationpathway(s) mediated by these receptors is still unknown. Ithas been shown, however, that spores contain different typesof receptors that can respond to specific germinant molecules,mostly amino acids or purine ribonucleosides (1, 4, 10, 11,13, 19, 27). Germination receptors are encoded by tricistronicoperons called ger operons, and the three gene products arenecessary to form a functional receptor (19). During sporulation,the ger operons are transcribed in the forespore by a sigmaG-dependent RNA polymerase (21). The genome of B. cereus ATCC14579 contains seven putative ger operons, which may equip thespore with a set of seven functional receptors (11). The Bacillussubtilis genome contains five ger operons, of which three havebeen characterized (5, 14, 28). These three ger operons wereexpressed at very low levels (5, 8, 28). The composition ofthe medium can affect various spore properties (3, 7, 9), butthe effect of medium composition on the transcription of theger operons and its impact on germination properties of thespores is not known. Changes in ger operon expression may causevariation in the number of receptors in the spore, which consequentlyaffects the nutrient-induced germination properties.

This report describes the transcriptional analysis of each ofthe seven ger operons of B. cereus ATCC 14579 during sporulationin nutrient-rich Y1 medium, containing approximately 30 mM aminoacids and 10 mM glucose, and in modified G medium, containingapproximately 14 mM amino acids and no glucose. The compositionof the medium had a significant impact on expression of theB. cereus ger operons and the spores' nutrient-induced germinationcharacteristics.

Bacterial strains, spore preparation, and transcriptional analysis.

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Bacterial strains, spore...

The B. cereus ATCC 14579 ger mutant strains used were disruptedin each of the seven ger operons by the insertion of plasmidpMUTIN4 as described previously (11). For all insertions, thelacZ reporter gene present on pMUTIN4 was under the controlof the ger operon promoter, facilitating the measurement oftranscriptional activity under different sporulation conditions.Spores of the wild-type and mutant strains were prepared ona nutrient-rich, chemically defined sporulation medium designatedY1 medium, which contained the following components (final concentrations):D-glucose (10 mM), L-glutamic acid (20 mM), L-leucine (6 mM),L-valine (2.6 mM), L-threonine (1.4 mM), L-methionine (0.47mM), L-histidine (0.32 mM), sodium-DL-lactate (5 mM), aceticacid (1 mM), FeCl₃ (50 µM), CuCl₂ (2.5 µM), ZnCl₂(12.5 µM), MnSO₄ (66 µM), MgCl₂ (1 mM), (NH₄)₂SO₄(5 mM), Na₂MoO₄ (2.5 µM), CoCl₂ (2.5 µM), and Ca(NO₃)₂(1 mM). The medium was buffered at pH 7.2 with 100 mM potassiumphosphate buffer (6). Furthermore, spores were prepared on modifiedG medium as described previously (15); the medium contained0.2% yeast extract, CaCl₂ (0.17 mM), K₂HPO₄ (2.87 mM), MgSO₄(0.81 mM), MnSO₄ (0.24 mM), ZnCl₂ (17 µM), CuSO₄ (20 µM),FeCl₃ (1.8 µM), and (NH₄) ₂SO₄ (15.5 mM) and was adjustedto a pH of 7.2. This medium was expected to contain approximately14 mM amino acids, based on a 70% protein content of the yeastextract. Cultures were incubated at 30°C with shaking at225 rpm, which resulted in >99% free spores in both media,after incubation for 48 h. The spores were then harvested, washedrepeatedly, and stored as described previously (11).

Transcriptional activity of the ger operons during sporulationwas measured by determining the level of ß-galactosidaseactivity using the 4-methylumbelliferyl-ß-D-galactoside(MUG) assay. One-milliliter samples of a sporulating wild-typeor mutant culture were taken at 5, 10, 15, 20, and 25 h afterinoculation and washed and stored at –20°C until assayed.The ß-galactosidase activity was assayed by measuringthe fluorescence that resulted from the conversion of MUG to4-methylumbelliferone with a Tecan fluorometer as describedpreviously (12). The instrument was calibrated with a 4-methylumbelliferonecalibration curve. Spore germination was monitored by measuringthe reduction of the optical density at 600 nm (OD₆₀₀) of thespore suspension as described previously (10). The data presentedare the result of three independent experiments.

Growth and sporulation in Y1 and modified G sporulation media.

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Growth and Sporulation in...

In both media, vegetative growth was observed first before thecells entered sporulation. After 4 h of vegetative growth inmodified G medium, at which point a maximum cell density of1.5 (OD₆₀₀) was reached, the cells entered sporulation. ForY1 medium, vegetative growth was observed for 12 h, at whichpoint a maximum cell density was reached (OD₆₀₀, 4.5), and thecells entered sporulation. The B. cereus ATCC 14579 wild typeand seven ger mutants displayed similar growth characteristics,including growth rates, final ODs, and spore yields during sporulation.The shift from growth to sporulation was accompanied by strongaggregation of the cells. Sporulation in the two media was synchronousand complete.

Transcription levels of the ger operons in Y1 and modified G sporulation media.

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Transcription levels of the...

Transcription levels of the ger operons were determined by measuringthe amount of ß-galactosidase produced in each ofthe seven ger mutant strains at five time points during sporulation.Transcription of the ger operons reached a maximum approximately10 h after the start of sporulation. At 20 h after the onsetof sporulation, the maturing spores became increasingly insensitiveto lysozyme treatment. Because the onset of sporulation in thetwo media started at different time points, a mutual comparisonof ß-galactosidase levels of samples taken at identicaltime points was not possible. Therefore, we compared the maximumexpression levels of each ger operon of sporulating B. cereuscells in both media. In Y1 medium, the maximum expression levelsof the ger operons during sporulation were substantially higherthan in modified G medium (Fig. 1). On average, expression ofthe seven ger operons was 3.5 times higher per cell in Y1 mediumthan in spores produced in modified G medium. Furthermore, differencesin expression levels for the single ger operons were noticed,although in both media, the highest transcription level wasobserved for the gerI operon encoding a germination receptorinvolved in purine ribonucleoside-initiated germination (4,11). Although the mutants harbor the same promoterless lacZgene, diversity in the ribosome binding sites and translationstart codons of the ger mutants may influence the ß-galactosidaselevels originating from these constructs. Therefore, we cannotbe sure that the relative levels of ß-galactosidaseare a precise reflection of the relative levels of the Ger receptors.A comparison of the levels of ß-galactosidase producedin the two media was done with the same strains, and this reflectsa valid comparison of the ß-galactosidase levels producedin the two media.

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FIG. 1. Relative ß-galactosidase activity expressed from the ger-lacZ fusions constructed for the seven ger operons of B. cereus during sporulation on modified G (black bars) and Y1 (gray bars) sporulation media. Maximum ß-galactosidase activity was measured 10 h after the onset of sporulation in the respective medium. The number of enzyme units was calculated per ml of culture and corrected for the observed difference in cell density. Data are expressed as a percentage of the maximum value obtained with the gerI-lacZ construct (20 pmol MUG hydrolyzed per ml of culture). WT, wild type.

Germination characteristics of spores produced on Y1 and modified G media.

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Germination characteristics of...

To assess the germination characteristics of spores preparedin both media, spore germination assays were performed usingthe amino acids L-alanine, L-cysteine, and L-threonine and thepurine ribonucleosides inosine and adenosine as germinant molecules.For both amino acid- and purine ribonucleoside-induced germination,spores prepared on modified G medium exhibited altered germinationcharacteristics compared to spores produced on Y1 medium. Thiswas demonstrated by reduced germination rates for amino acid-inducedgermination (Fig. 2) and a reduced germination rate and a morepronounced lag phase for purine nucleoside-induced germination(Fig. 3) for spores grown on modified G medium. Spores frommodified G medium required, on average, 10-fold higher concentrationsof germinants to accomplish germination rates and efficienciessimilar to those of Y1-medium-grown spores (data not shown).By adding a synergistic combination of L-alanine and inosine,resulting in a powerful germinant mixture, spores originatingfrom both media showed immediate responses and high, almostsimilar, germination rates (Fig. 3). This indicates that a potentgermination trigger may overcome a limited number of nutrientreceptors. To analyze whether these differences in spore germinationcharacteristics resulted from an early (stage 1) germinationevent (24), both types of spores were germinated with Ca²⁺-dipicolinicacid. This nonnutrient germinant induces germination while bypassingthe individual components (i.e., the Ger receptors) of the nutrientgermination pathway. Ca²⁺-dipicolinic acid-induced germinationshowed equal rates and efficiencies with both types of spores,indicating that the germination pathways after stage 1 are similarin spores originating from Y1 and modified G media (data notshown). This observation points toward a difference in the firststage of germination, and based on the transcriptional analysisof all B. cereus ATCC 14579 ger operons, it is conceivable thatthe number of germination receptors is lower in spores preparedon modified G medium, resulting in reduced germination ratesand efficiencies.

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FIG. 2. Germination of B. cereus wild-type spores produced on Y1 (closed symbols) and modified G (open symbols) media. Spores were germinated with 1 mM L-alanine (circles), L-cysteine (squares), and L-threonine (triangles).

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FIG. 3. Germination of B. cereus wild-type spores produced on Y1 (closed symbols) and modified G (open symbols) media. Spores were germinated with a combination of 10 mM L-alanine and 1 mM inosine (circles), with 1 mM inosine alone (squares), and with 1 mM adenosine (triangles) alone.

It has been described previously that the level of ger operontranscription can affect germination characteristics. Overexpressionof the gerA operon in B. subtilis, which elevates the levelof the GerA proteins, resulted in a more rapid response forL-alanine-induced germination (2). Therefore, the lower numberof germination receptors that result from sporulation in modifiedG medium may result in a diminished germination rate.

For purine ribonucleoside-induced germination (Fig. 3), thelow levels of expression of the ger operons measured in modifiedG medium-grown spores correlated with a prolonged lag phasebefore germination started. Once started, the germination ratesand efficiencies were only slightly lower for the modified Gmedium-produced spores. As discussed previously, the amino acid-inducedgermination seems to proceed via a partially different pathwaythan the purine ribonucleoside-induced germination (11, 25).It is known that the integument and exosporium of the sporeharbor active enzymes, including inosine hydrolase, that mayconvert compounds added to the spore suspension, such as purineribonucleosides, into effective germinants (16, 17, 23). Suchenzymes may play an essential role in purine ribonucleoside-inducedgermination, and the observed lag phase may be caused by thetime needed to convert adenosine or inosine into effective concentrationsof the actual germinant molecule(s). This resulting moleculemay initiate germination mediated by, for example, the GerIreceptor, which has been shown to be involved in the inosineand adenosine responses in B. cereus ATCC 14579 (1, 11). Assumingthat lower numbers of receptors are present in spores originatingfrom modified G medium, a higher concentration of the actualgerminant molecule may be required before germination can proceed.Consequently, more time is needed to produce a sufficient quantityof this germinant molecule, resulting in the observed prolongedlag phase. For amino acid-induced germination, the germinationrate correlates with the concentration of the germinant (10),and the assumption that spores produced on modified G mediumcontain a lower number of receptors may be reflected directlyin the germination rate.

In the environment, sporulation conditions can be extremelydiverse, and properties of the resulting spores, including germinationefficiencies, may differ significantly. This large diversityin spore properties may add to the success of spore dispersalin foods and affect spore survival and outgrowth potential afterthe imposition of food preservation technologies.

FOOTNOTES

* Corresponding author. Mailing address: Laboratory of Food Microbiology, Wageningen University, Bomenweg 2, 6703 HD Wageningen, The Netherlands. Phone: 31-317-484981. Fax: 31-317-484978. E-mail: tjakko.abee{at}wur.nl.

REFERENCES

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